Not applicable.
The invention provides isolated nucleic acid and amino acid sequences of hElk, antibodies to hElk, methods of detecting hElk, methods of screening for voltage-gated potassium channel activators and inhibitors using biologically active hElk, and kits for screening for activators and inhibitors of voltage gated potassium channels comprising hElk.
Potassium channels are involved in a number of physiological processes, including regulation of heartbeat, dilation of arteries, release of insulin, excitability of nerve cells, and regulation of renal electrolyte transport. Potassium channels are thus found in a wide variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7):805-829 (1997)). Three of these families (Kv, Eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families, CNG and SK/IK, also contain this motif but are gated by cyclic nucleotides and calcium, respectively. The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066-71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273:3509-16 (1998)). Another family, the inward rectifier potassium channels (Kir), belong to a structural family containing 2 transmembrane domains (see, e.g., Lagrutta et al., Jpn. Heart. J. 37:651-660 1996)), and an eighth functionally diverse family (TP, or xe2x80x9ctwo-porexe2x80x9d) contains 2 tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These beta subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493:625-633 (1996); Shi et al., Neuron 16(4):843-852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384:80-83 (1996)).
The Kv superfamily of voltage-gated potassium channels includes both heteromeric and homomeric channels that are typically composed of four subunits. Voltage-gated potassium channels have been found in a wide variety of tissues and cell types and are involved in processes such as neuronal integration, cardiac pacemaking, muscle contraction, hormone section, cell volume regulation, lymphocyte differentiation, and cell proliferation (see, e.g., Salinas et al., J. Biol. Chem. 39:24371-24379 (1997)).
A family of voltage-gated potassium genes, known as the xe2x80x9cEagxe2x80x9d or ether à go-go family, was identified on the basis of a Drosophila behavioral mutation with a leg-shaking phenotype (see, e.g., Warmke and Ganetzky, Proc. Nat""l Acad. Sci. USA 91:3438-3442 (1994)). Family members from Drosophila and vertebrates have been cloned and fall into three subfamilies. One such subfamily is called the Eag subfamily and is represented, e.g., by Drosophila Eag (Warmke et al., Science 252:1560-1562 (1991); Bruggemann et al., Nature 365:445-447 (1993)), and rat, mouse, human, and bovine Eags (Ludwig et al., EMBO J. 13:4451-4458 (1994); Robertson et al. Neuropharmacology 35:841-850 (1996); Occhiodoro et al., FEBS Letters 434:177-182 (1998); Shi et al., J. Physiol. 115.3:675-682 (1998); Frings et al., J. Gen Physiol. 111:583-599 (1998)). A second subfamily, the Erg or xe2x80x9cEag-related genexe2x80x9d family is represented, e.g., by human erg (Shi et al., J. Neurosci. 17:9423-9432 (1997)). Finally, a third subfamily, the Elk or xe2x80x9cEag-like K+ genexe2x80x9d is represented, e.g., by Drosophila Elk (Warmke et al., Proc. Natl. Acad. Sci. 91:3438-3442 (1994)).
The present invention thus provides for the first time human Elk, a polypeptide monomer that is an alpha subunit of an voltage-gated potassium channel. hElk has not been previously cloned or identified, and the present invention provides the nucleotide and amino acid sequence of hElk. Furthermore, the gene encoding hElk has been mapped to chromosome 12.
In one aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Elk alpha subunit, a potassium channel having the characteristic of voltage gating; (ii) having a monomer P-S6 region that has greater than 80%, preferably 85, 90, or 95% amino acid sequence identity to an hElk P-S6 region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1.
In another embodiment, the present invention provides an isolated nucleic acid encoding a polypeptide monomer comprising an alpha subunit of a potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Elk alpha subunit, a potassium channel having the characteristic of voltage gating; (ii) having an extended P-S6 region that has greater than 80%, preferably 85, 90, or 95% amino acid sequence identity to an hElk extended P-S6 region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1.
In one embodiment, the nucleic acid encodes human Elk. In another embodiment, the nucleic acid encodes SEQ ID NO:1. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:2.
In one embodiment, the nucleic acid is amplified by primers that selectively hybridize under stringent hybridization conditions to the same sequence as the primer sets selected from the group consisting of: ATGCCGGCCATGCGGGGCCTCCT (SEQ ID NO:3), AGATGGCAGCACACCTGGCAACGCTG (SEQ ID NO:4) and GCCCATCTGCTGAAGACGGTGCGC (SEQ ID NO:5), CGAAGCCCACGCTGGTGAGGCTGCTG (SEQ ID NO:6).
In one embodiment, the nucleic acid encodes a polypeptide monomer having a molecular weight of between about 120 kDa to about 130 kDa. In another embodiment, the polypeptide monomer comprises an alpha subunit of a homomeric voltage-gated potassium channel. In another embodiment, the polypeptide monomer comprises an alpha subunit of a heteromeric voltage-gated potassium channel.
In another embodiment, the nucleic acid selectively hybridizes under moderately stringent hybridization conditions to a nucleotide sequence of SEQ ID NO:2.
In another aspect, the present invention provides an isolated nucleic acid encoding a polypeptide monomer, wherein the nucleic acid specifically hybridizes under highly stringent conditions to SEQ ID NO:2.
In another aspect, the present invention provides an isolated polypeptide monomer comprising an alpha subunit of a potassium channel, the polypeptide monomer: (i) having the ability to form, with at least one additional Elk alpha subunit a potassium channel having the characteristic of voltage gating; (ii) having a monomer P-S6 region that has greater than 80% amino acid sequence identity to an hElk P-S6 region; and (iii) specifically binding to polyclonal antibodies generated against SEQ ID NO:1.
In one embodiment, the polypeptide monomer has an amino acid sequence of human Elk. In another embodiment, the polypeptide monomer has an amino acid sequence of SEQ ID NO:1.
In another aspect, the present invention provides an antibody that selectively binds to the polypeptide monomer described above.
In another aspect, the present invention provides an expression vector comprising the nucleic acid encoding the polypeptide monomer described above.
In another aspect, the present invention provides a host cell transfected with the expression vector described above.
In another aspect, the present invention provides a method for identifying a compound that increases or decreases ion flux through an voltage-gated potassium channel, the method comprising the steps of: (i) contacting the compound with a eukaryotic host cell or cell membrane in which has been expressed a polypeptide monomer comprising an alpha subunit of a potassium channel, the polypeptide monomer: (a) having the ability to form, with at least one additional Elk alpha subunit, a potassium channel having the characteristic of voltage gating; (b) having a monomer P-S6 region that has greater than 80% amino acid sequence identity to an hElk P-S6 region; and (c) specifically binding to polyclonal antibodies generated against SEQ ID NO:1; and (ii) determining the functional effect of the compound upon the cell or cell membrane expressing the potassium channel.
In one embodiment, the increased or decreased flux of ions is determined by measuring changes in current or voltage. In another embodiment, the polypeptide monomer polypeptide is recombinant. In another embodiment, the potassium channel is homomeric.
In another embodiment, the present invention provides a method of detecting the presence of hElk in human tissue, the method comprising the steps of: (i) isolating a biological sample; (ii) contacting the biological sample with a hElk-specific reagent that selectively associates with hElk; and, (iii) detecting the level of hElk-specific reagent that selectively associates with the sample.
In one embodiment, the hElk-specific reagent is selected from the group consisting of: hElk specific antibodies, hElk specific oligonucleotide primers, and hElk nucleic acid probes.
In another aspect, the present invention provides, in a computer system, a method of screening for mutations of hElk genes, the method comprising the steps of: (i) entering into the computer a first nucleic acid sequence encoding an voltage-gated potassium channel protein having a nucleotide sequence of SEQ ID NO:2, and conservatively modified versions thereof; (ii) comparing the first nucleic acid sequence with a second nucleic acid sequence having substantial identity to the first nucleic acid sequence; and (iii) identifying nucleotide differences between the first and second nucleic acid sequences.
In one embodiment, the second nucleic acid sequence is associated with a disease state.
In another aspect, the present invention provides, in a computer system, a method for identifying a three-dimensional structure of hElk polypeptides, the method comprising the steps of: (i) entering into the computer an amino acid sequence of at least 25, 50 or 100 amino acids of a potassium channel monomer or at least 75, 150 or 300 nucleotides of a nucleic acid encoding the polypeptide, the polypeptide having an amino acid sequence of SEQ ID NO:1, and conservatively modified versions thereof; and (ii) generating a three-dimensional structure of the polypeptide encoded by the amino acid sequence.
In one embodiment, the amino acid sequence is a primary structure and wherein said generating step includes the steps of: (i) forming a secondary structure from said primary structure using energy terms determined by the primary structure; and (ii) forming a tertiary structure from said secondary structure using energy terms determined by said secondary structure. In another embodiment, the generating step includes the step of forming a quaternary structure from said tertiary structure using anisotropic terms determined by the tertiary structure. In another embodiment, the methods further comprises the step of identifying regions of the three-dimensional structure of the protein that bind to ligands and using the regions to identify ligands that bind to the protein.